Embryonic stem cells (ES cells) are stem cells established from human or mouse early embryos which have a characteristic feature that they can be cultured over a long period of time while maintaining pluripotent ability to differentiate into all kinds of cells existing in living bodies. Human embryonic stem cells are expected for use as resources for cell transplantation therapies for various diseases such as Parkinson's disease, juvenile diabetes, and leukemia, taking advantage of the aforementioned properties. However, transplantation of ES cells has a problem of causing rejection in the same manner as organ transplantation. Moreover, from an ethical viewpoint, there are many dissenting opinions against the use of ES cells which are established by destroying human embryos.
If dedifferentiation of patients' own differentiated somatic cells could be induced to establish cells having pluripotency and growth ability similar to those of ES cells (these cells are referred herein to as “induced pluripotent stem cells” or “iPS cells,” though they are sometimes called “embryonic stem cell-like cells” or “ES-like cells”), it is expected that such cells would be useful as ideal pluripotent cells, free from rejection or ethical difficulties. Recently, it has been reported that such iPS cells can be produced from differentiated cells of mouse or human, which has created a great sensation (International Publication No. WO2007/069666 A1; Takahashi et al., Cell 126:663-76, 2006; Takahashi et al., Cell 131:861-72, 2007; Yu et al., Science 318:1917-20, 2007; and Park et al., Nature 451:141-46, 2008, herein incorporated by reference in their entireties). Thus, the term “induced pluripotent stem cells (iPS cells)” refers to cells having similar properties to those of ES cells, and more specifically the term includes undifferentiated cells which are reprogrammed from somatic cells and have pluripotency and proliferation potency. However, this term is not to be construed as limiting in any sense, and should be construed to have its broadest meaning.
These methods include a reprogramming step through introduction of a plurality of specific factors (for example, four factors of Oct3/4, Sox2, Klf4, and c-Myc can be used in Takahashi et al., Cell 126:663-76, 2006), and the introduction of these factors is mediated by viral vectors such as retroviral or lentiviral vectors. However, all previously reported nuclear reprogramming methods mediated by the introduction of genes involve a problem of low efficiency in which only a small number of induced pluripotent stem cells can be obtained. In particular, there is a problem in that, if reprogramming is carried out in somatic cells through the introduction of three factors (namely, Oct3/4, Sox2, and Klf4) excluding c-Myc, then the production efficiency of induced pluripotent stem cells becomes low. Nevertheless, the efficient production of iPS cells without the use of c-Myc would provide certain advantages, as c-Myc is suspected to cause tumorigenesis in tissues and in chimeric mice generated from induced pluripotent stem cells.
It is known that various small RNAs are expressed in cells. Examples of small RNA include RNA molecules of about 18-25 nucleotides in length which can be cleaved out with a dicer, an RNase specific to double-stranded RNA. Small RNA is mainly classified into siRNA (small interfering RNA) and miRNA (microRNA, hereinafter abbreviated as “miRNA”). Small RNA is known to function as a guide molecule for finding target sequences in processes such as translational suppression, mRNA degradation, or alteration of chromatin structure. Small RNAs function via RNA interference (RNAi) or miRNA molecular mechanisms. In addition, small RNA is also known to play an important role in the regulation of developmental processes (for example, as general remarks, refer to Jikken Igaku (Experimental Medicine), 24, pp. 814-819, 2006; and microRNA Jikken Purotokoru (microRNA Experimental Protocol), pp. 20-35, 2008, YODOSHA CO., LTD., herein incorporated by reference in their entireties).
ES cell-specific microRNAs have been identified (Houbaviy et al., Developmental Cell 5:351-58, 2003). In particular, ES cell-specific expression of a microRNA cluster, which includes several types of miRNAs in mouse ES cells, has been reported (Houbaviy et al., Developmental Cell 5:351-58, 2003, herein incorporated by reference in its entirety). It has also been reported that miRNA-295 suppressed the expression of Rbl2, a member of the Rb tumor suppressor gene family, and increased the expression of methylase to be thereby associated with DNA methylation (Sinkkonen et al., Nature Structural & Molecular Biology 15:259-267, 2008; Benetti et. al., Nature Structural & Molecular Biology 15:268-279, 2008, herein incorporated by reference in their entireties). However, these documents do not disclose any role of small RNA in the nuclear reprogramming of somatic cells.